Turn-on Fluorescent Biosensors for Imaging Hypoxia-like Conditions in Living Cells

We present the synthesis, photophysical properties, and biological application of nontoxic 3-azo-conjugated BODIPY dyes as masked fluorescent biosensors of hypoxia-like conditions. The synthetic methodology is based on an operationally simple N=N bond-forming protocol, followed by a Suzuki coupling, that allows for a direct access to simple and underexplored 3-azo-substituted BODIPY. These dyes can turn on their emission properties under both chemical and biological reductive conditions, including bacterial and human azoreductases, which trigger the azo bond cleavage, leading to fluorescent 3-amino-BODIPY. We have also developed a practical enzymatic protocol, using an immobilized bacterial azoreductase that allows for the evaluation of these azo-based probes and can be used as a model for the less accessible and expensive human reductase NQO1. Quantum mechanical calculations uncover the restructuration of the topography of the S1 potential energy surface following the reduction of the azo moiety and rationalize the fluorescent quenching event through the mapping of an unprecedented pathway. Fluorescent microscopy experiments show that these azos can be used to visualize hypoxia-like conditions within living cells.


EXPERIMENTAL PROCEDURES
Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich, Fluorochem and Alfa Aesar Chemicals and were used without further purification. Air sensitive reactions were performed using oven-dried glassware (Oven = 150 °C) and performed under a nitrogen atmosphere using Schlenk techniques. Solvents were dried on a solvent purification system (PS-MD-5/7 Inert technology). Reactions were monitored by thin-layer chromatography (TLC) on silica-gel-coated aluminum foils (silica gel 60 F254, Merck). The TLC plates were visualized by UV light (λ = 254 nm). Flash-column chromatography was performed on silica gel 60. NMR spectra were recorded on a Bruker AV-300 spectrometer at 25 °C. Chemical shifts (δ) are reported in ppm relative to the residual solvent peak. Splitting patterns are indicated as (s) singlet, (d) doublet, (t) triplet, (q) quartet, (m) multiplet and (br) broad. Coupling constants (J) are reported in Hertz (Hz). High-resolution mass spectra (HRMS) were recorded on a Waters XEVO-G2 QTOF mass spectrometer, using electron impact (EI) or electrospray ionization (ESI) in positive or negative mode, depending on the analyte. UV-Visible experiments were conducted using a JASCO V-660 apparatus at room temperature. Emission spectra were recorded in a JASCO FP-8600 equipment. Quartz cuvettes were used for the measurements, in particular Hellma® precision cells made of Quartz Suprasil ® (10 x 10 mm). In these two instruments the temperature was controlled using a JASCO Peltier thermostated cell holder with a range of 263-383 K, adjustable temperature slope, and accuracy of ± 0.1 K. Solvents were purified by use of drying cartridges through a solvent delivery system. All chemicals were used as received unless otherwise noted. BODIPY 1 1 and 4 2 were synthesized according to the literature. Bis-dimethyl acetal of pbenzoquinone 2 is commercially available and can also be synthesized according to the literature. 3

SYNTHESIS OF STARTING MATERIALS.
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Synthesis of 2,5-Dichloro-8-phenyl-BODIPY 2b
To a two necked 250 mL round bottom flask equipped with a stir bar was added 5-phenyl-dipyrromethane 4 (1.0 g, 4.5 mmol, 1.0 equiv.), the flask was evacuated and flushed with argon three times before 150 mL of dry tetrahydrofuran (0.03M) was added. The solution was cooled to -78 °C before N-chlorosuccinimide (2.5 equiv.) was added under an argon flow in one portion. The reaction mixture was stirred at -78 °C for 2 h and at -20 °C overnight. The reaction mixture was diluted with water and extracted with DCM. The combined organic solvent was washed with brine, dried over MgSO4 and concentrated under reduce pressure. The obtained residue was placed in a 250 mL round-bottom flask and p-chloranil (1.1 g, 4.5 mmol, 1.0 equiv.) was added. Then, under an argon atmosphere dry dichloromethane (0.03 M) was added and the resulting reaction mixture stirred at rt for 2 h. Then, DIPEA (5.8 g, 7.9 mL, 45 mmol, 10 equiv.) was added at 0 ºC, after 10 min BF3·OEt2 (19.2 g, 16.6 mL, 135 mmol, 30 equiv.) was added also at 0 ºC. The reaction was allowed to warm to rt and stirred for 2 h. Then, saturated NaHCO3 (30 mL) was slowly added to the crude mixture at 0 °C and extracted with DCM (30 ml x 3). The combined organic layers were dried over MgSO4, the solvent was concentrated under reduce pressure and residue was purified by flash column chromatography using heptane: CH2Cl2 (1.5:1) as eluent to afford 4 as bright red powder (1 g, 67% yield). 1 H NMR (300 MHz, CDCl3): δ = 7.70-7.43 (m, H), 6.95 (d, J = 4.3 Hz, 2H), 6.81 (d, J = 4.3 Hz, 2H).

Synthesis of bis-dimethyl acetal of p-benzoquinone (3,3,6,6-tetramethoxycyclohexa-1,4-diene) 2 3
A solution of 1,4-dimethoxybenzene (17 g, 123 mmol) and KOH in 200 mL of MeOH was anodically oxidized at -5 °C, under constant current (1.0 A, 2 V), [Electrolysis was carried out in a single cell apparatus, using a circular platinum gauze anode (5 cm x 5 cm in diameter) 45-mesh, a graphite cathode (9 mm x 40 mm), and an AMEL (model 549) power supply]. The reaction was monitored by TLC. When the reaction was completed, the methanolic solution was evaporated in vacuo to afford a pale orange paste. This material was dissolved in ca. 200 ml of hot petroleum ether, filtered, and washed with water and saturated brine solution. The combined organic phases were dried over anhydrous MgSO4, filtered and concentrated in vacuo, affording a light yellow solid which was recrystallized from petroleum ether to yield 2 as white crystals in 76% yield (18.6 g, 98.89 mmol). 1 H NMR (300 MHz, CDCl3): δ = 6.10 (s, 4H), 3.30 (s, 12H).

One pot synthesis of 5-chloro-3-arylazo-BODIPY 3 3-Chloro-5-(4-methoxyphenyl)diazenyl-8-phenyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene 3
3,5-Dichloro BODIPY 4 (1.0 g, 3.0 mmol) was added to an oven dried microwave tube; the tube was sealed, evacuated and flushed with argon three times before dry acetonitrile (0.03 M) and N2H4 . H2O (2.0 equiv) were added. Then, the resulting solution was heated at 80 ºC for 2 h. After that period of time the reaction mixture was cooled to room temperature and the tube was opened before 1,1,4,4-tetramethoxycyclohexa-1,4-diene 2 (2.0 equiv) and CAN (10 mol %) were added (after the addition of CAN, a fast and drastic change of color from dark red to deep dark blue was observed). Finally, when the reaction was finished (monitored by TLC), the solvent was evaporated under reduce pressure and the residue was purified by flash column chromatography using Heptane: CH2Cl2 (1:1) as eluent to afford the desire product as a dark powder in 73% yield (967 mg, 2.2 mmol). 4.2 General method A: Synthesis of 5-Aryl (heteroaryl)-3-arylazo BODIPY 5a-i General procedure A: To an oven dried microwave tube was added 5-chloro-arylazo BODIPY 3 (100 mg, 0.23 mmol, 1.0 equiv), the corresponding boronic acid acid (2.0 equiv), Pd(OAc)2 (5 mol%), P(2-OMe-Ph)3 (10 mol%) and Na2CO3 (3.0 equiv). The tube was evacuated and flushed with argon three times before dry dimethoxyethane (0.05 M) was added. The resulting suspension was heated at 60 ºC for 2 h. Then, the reaction mixture was diluted with CH2Cl2 and filtered in short pad of celite. After that time the solvent was evaporated in vacuo and the residue was purified by flash column chromatography using a gradient of Heptane: CH2Cl2 from [2:1] to [1:1] as eluent to afford the desire product as a dark powder.

Synthesis of 3-amino-5-aryl(heteroaryl)-BODIPY 5a-i
To a suspension of the corresponding azo-BODIPY 5 (1.0 equiv) and zinc dust (10 equiv) in CH2Cl2 (0,1 M), ammonium formate (20 equiv), dissolved in the minimum amount of methanol, was added and the mixture was stirred at room temperature. After the completion of the reaction (monitored by TLC) the reaction mixture was filtered through a pad of Celite, the solvent was evaporated in vacuo and the residue was purified by flash column chromatography using heptane:AcOEt as eluent to afford the desire product.

UV-Vis/EMISSION SPECTRA
Absorbance spectrum of azo BODIPY 5a-i and absorbance and emission spectrum of amino BODIPY 6a-i.

Fluorescence Quantum Yields
Quantum yields were determined by measuring both absorbance and fluorescence of amino-BODIPY 6 and Rhodamina 101 in EtOH (Rhodamina 101 as standard r = 1). 5 The measurements were performed using 10×10 mm cuvettes on non-degassed samples. Quantum yields were determined in DCM for all amino-BODIPY 6 and also in DMSO/PBS for 6i. For the relative determination of the fluorescence quantum yield (), the following formula was used: 6 S18 Subscripts x and r refer to sample and reference (standard) fluorophore respectively with known quantum yield r in a specific solvent F stands for the spectrally corrected, integrated fluorescence spectra. A(λex) denotes the absorbance at the used excitation wavelength λex. n represents the refractive index of the solvent (at the average emission wavelength).
To minimize inner filter effects, the absorbance at the excitation wavelength λex was kept under 0.1.

Computational details
Ground state geometry optimizations for the azo-BODIPY 5i and the amino-BODIPY 6i were performed at the wB97X-D/cc-pVDZ 7,8 level of theory, in a continuum of dichloromethane under the Polarizable Continuum Model (PCM) using the integral equation formalism variant (IEFPCM). 9,10,11 Subsequent frequency calculations to check the nature of the stationary points found were undertaken at the same level of theory. These calculations were done with the Gaussian 16 (Revision C.01). 12 The rotational flexibility of the 5i derivative requires a conformational analysis to identify the most stable conformer(s) at thermal equilibrium, for which we will model the photophysics. The most stable conformers differing in the orientation of the thiophene and azo groups relative to the BODIPY moiety were scrutinized. In turn, the effect on the internal energy of the rotation of the methoxy substituent around the CArO bond was also evaluated. The geometries, relative energies in solution and population according to a Boltzmann distribution at 298 K of the 8 most stable conformers are collected in Figure S4. The two most stable species (conformers D, 51% and C, 36%) have in common the layout of the three chromophores while differ in the dihedral of the methoxy group. Since the rotation of the OCH3 substituent around the CArO bond is not expected to significantly change the ground and excited potential energy landscape, all the subsequent electronic structure calculations were performed on the major D conformer. For the D conformer of 5i and 6i, gas phase vertical excitation energies were calculated at the RI-ADC(2)/def2-SVP 13,14,15,16,17,18 level of theory with Turbomole 7.02. 19 Natural transition orbitals (NTOs) and excitation analysis was carried out with the TheoDORE package. 20 Excited state geometry optimizations for minima, transition states and conical intersections were undertaken at the RI-ADC(2)/def2-SV(P) level of theory, using in the latter case SHARC 21,22 as an interface between Turbomole, for energy and gradient calculations, and Orca 4.1, as a geometry optimizer. 23 Additionally, for benchmarking purposes, gas phase S1 vertical excitation energies for azo-BODIPY 5i and for amino-BODIPY 6i were also respectively computed at the MS3-CASPT2(8,6)/ANO-S-VDZ and MS5-CASPT2(12,10)/ANO-S-VDZ levels of theory with the OpenMolcas code. 24 For the 5i derivative, the CASPT2 S1 excitation energy of 1.91 eV (640 nm) is in excellent agreement with the experimental absorption spectrum (585 nm, see Table S2). Additionally, a good agreement between the CASPT2 and the ADC(2) levels of theory was found for the S1 excited state energy (∆E~0.3 eV). It is particularly interesting that the state order is kept at both levels of theory, being the S1 (ππ*) the first bright state, S2 (nπ*) the dark state and S3 (ππ*) the second bright state. According to the CASPT2 approach, the S1 state in 6i absorbs at 2.29 eV (541 nm), also in good agreement with both experimental data and ADC(2) calculations.

Reactive ground and excited PES for azo-BODIPY 5i
For the 5i derivative, three different ground state minima were found: trans, cis bottom and cis top, with relative energies 0, 0.51 and 0.51 eV, respectively (see Figure S5). The two cis isomers differ in the relative orientation of the azobenzene moiety with respect to the BODIPY resulting in positive (cis bottom) or negative (cis top)

Conformers TRANS
A B **There are more posible conformers.

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helicities. These minima are connected to the trans isomer through energy barriers of ~1.4 eV (~32 Kcal/mol) which should avoid fast thermal isomerization at room temperature. These transition states (TS top and TS bottom , see  Figures S5 and S9) show geometries close to those of the reactive S1/ S0 conical intersections: CI top -S1(ππ*)/S0 and CI bottom -S1(ππ*)/S0. Figure S5 shows both possible reactive pathways starting from the trans isomer, and evolving either towards the cis top or the cis bottom isomer. For clarity, and due to the symmetry of the S1 and S0 potential energy surfaces along the isomerization coordinate, only the potential energy landscape connecting the trans and cis bottom isomer is shown in Figure 4 of the main manuscript. The unreactive pathway can be also found in Figure 4 (a) of the main manuscript.  Figure S6).

ENZYMATIC AZO BOND CLEAVAGE STUDIES
Purified human NAD(P)H: quinone oxidoreductase 1 (NQO1 human) 25 was obtained from Sigma Chem. Co (St Louis Mo) (Catalogue No. SRP6539). The bacterial azoreductase from Bacillus cereus was produced and purified in the laboratory from the gene azoRBC that contains the sequence encoding the enzyme and the sequence encoding a poly-His tail per enzyme subunit. This gene was supplied by Genescript, NJ. The enzymatic studies were carried out using a Spectrophotometer JASCO v730 (Tokyo, Japan) with a spectrophotometric cell provided with magnetic stirring.

BACTERIAL AZOREDUCTASE (azoRBC) a) Expression, production and purification of azoRBC from the microorganism bacillus cereus
The gene coding for the azoRBC enzyme was cloned into a plasmid pET28b to be expressed recombinantly in the E. coli BL21 (DE3) organism. The expression was carried out in a ZY autoinduction medium containing 30 µg/mL of kanamycin at 37 ºC for 16 h. The cells were harvesting by centrifugation at 4000 rpm for 30 min. Cell pellets were suspended in 50 mM sodium phosphate buffer, 150 mM NaCl, 5 mM Imidazole and 3 mM benzamidine, pH 7.4 and lysed by means of 10 seconds ON/OFF sonication cycles at an amplitude of 20 % in an ice-water bath. Then, the lysate was centrifuged for 30 minutes at 14000 rpm. The pellet was discarded, and the protein was purified by an imidazole gradient using an agarose column IDA-Ni 2+ . The poly-histidine domain present at the Nterminus of each sub-unit strongly adsorbs on the metal chelate. The native proteins were eluted with 100 mM imidazole. Then, the recombinant enzyme was eluted with sodium phosphate buffer (50 mM), NaCl (150 mM), and Imidazole (250 mM) ( Figure S11).

Figure S11
Azoreductase purification: The plasmid containing the gene encoding the enzyme was expressed in Escherichia coli BL21. To purify the enzyme, a plasmid was acquired with its gene to which 6 histidine residues had been added at the amino terminal. The enzyme was purified by selective adsorption on supports activated with metal chelates (agarose column IDA-Ni 2+ ).
Finally, the pure enzyme was dialyzed against 25 mM sodium phosphate pH 7 and stored at 4 °C. Protein concentration was determined by Pierce® BCA kit and protein purity was checked by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis) ( Figure S12).

Preparation of i-azoRBC: Enzymatic immobilization on agarose PEI
Bacterial azoRBC was immobilized on a solid support (agarose gel) coated with polyethyleneimine (PEI). The enzyme was immobilized by an adsorption method promoted by the ionic exchange between several carboxyl groups of each enzyme molecule and several ionized amino groups of a polyethyleneimine-coated agarose support. Thus, the highly hydrophilic environment surrounding each enzyme molecule can also protect it from other negative effects promoted by the presence of ethanol that is necessary to solubilize substrates and products. The immobilized enzyme on a solid support was used and filter from the reaction medium, retaining the biocatalyst in a porous filter as soon as the reaction was finished. In this manner, the immobilize enzyme avoided the generation of aggregates that inactivated the enzyme. Two milliliters of diluted azo-RBC (0.5 mg/mL) with 60 mL of TRIS buffer (25 mM in water, pH 7), was added 3 g of agarose 6B support (6%). The agarose support had the surface completely covered with polyethyleneimine (PEI) of 25000 Da molecular weight. The suspension was gently stirred to avoid breaking the particles from the solid support. The initial activity of the solution (before adding the support) was 0.18 Δ Abs /min using 50 µL of the enzyme solution. The disappearance of the activity of the supernatant indicates the percentage of enzyme that is adsorbed on the solid support. On the other hand, the activity of the suspension indicates the percentage of activity that the enzyme retains after its immobilization. As it can be seen in Figure S14, the enzyme is completely immobilized

Enzymatic Activity and Stability studies
The standard activity of bacterial reductase azoRBC has been measured as a free azoRBC, and also during the immobilization process. Likewise, the standard activity has been also measured of the immobilized enzyme i-azoRBC and the human NQO1 enzyme. In all cases, Methyl Red which is a fairly water soluble azo derivative, was used as a reference. The concentration of Methyl Red could be increased a tenfold factor establishing as the standard reductase activity assay 50 µL of a 1 mg/mL solution of Methyl Red.

a. ACTIVITY OF i-azoRBC
The activities of soluble (azoRBC) and immobilized azoreductase (i-azoRBC) were measured in fully aqueous solution (buffer solution) and in a 1:1 water/ethanol solution. The assay followed the standard reductase activity assay using fully aqueous conditions (2 mL of buffer solution) or 50% ethanol (1 mL of ethanol/1 mL of buffer solution) and Methyl Red as a reference. The immobilized enzyme was assayed by using a stirring bar in the spectrophotometric cell in order to maintain the immobilized derivative perfectly suspended in the reaction mixture. The comparative results are shown in the following table.

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The immobilized enzyme (i-azoRBC) was approximately six times more active in the presence of ethanol than the free enzyme. This result could not only be related to aggregation phenomena but could also be due to the direct effect of ethanol, which decreases the enzymatic activity of the free enzyme. The presence of PEI in the immobilized derivative can reduce the effective concentration of ethanol in the surroundings of each immobilized enzyme molecule.

b. ENZYMATIC STABILITY STUDIES of i-azoRBC
The stability of the immobilized azoreductase and the free enzyme ( Figure S15) was studied using 1 mL of the corresponding enzyme (free or immobilized) diluted with 5 mL of buffer solution and 5 mL of ethanol at pH 7.0 (25 mM TRIS buffer). At different times, samples (200 µL) of each of the suspensions were withdrawn and activities were measured and compared with those that were obtained in the absence of ethanol. The activity was measured according to the standard reductase activity assay (50 µL of a 1 mg/mL solution of Methyl Red). The immobilized enzyme is shown to be more stable to ethanol than the soluble enzyme without immobilization. . The absorbance of the reaction mixture was registered to ensure that it was maintained constant and no precipitation nor undesirable adsorptions of the substrates on. The reaction was initiated through the addition of 30 µL of 10 mM of NADH (nicotine adenine dinucleotide). The decrease in absorbance (λmax at 430 nm for Methyl Red and at ~600 nm for each azocompound) were proportional to the rate of reduction of the different azocompounds.
In the case of immobilize enzyme (i-azoRBC), after the total reduction of each substrate (final absorbance lower than 0.01), the reaction mixture was easily separated from the immobilized enzyme by using a micro-filter syringe.
Since, azo-BODIPY compounds 5 were more soluble in the presence of 50% ethanol, and hence, higher substrate concentration could be used, the color changes associated with the reduction reaction products and the fluorescence of the different reaction products in 50% ethanol could be observed. The reaction mixture was placed in a fluorescent cell and illuminated with a deuterium lamp in the dark. The different azo-BODIPY were transformed into distinct fluorophores with pink, violent, or orange fluorescence. Representative examples of the products emitting a more intense fluorescence are shown in the following Table S5.

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(a) From the emission spectra spectra measurements described in Table 3 of the manuscript.
The reaction temperature was 25°C. The absorbance of the reaction mixture was constant before adding NADH. The rate of reduction of the azo-BODIPY was measured by the decrease in absorbance at the maximun λabs of each azo-BODIPY per minute. The reduction of Methyl Red is considered as 100% and the reduction rates of the different azo compounds are relative to it, results are shown in Table S6.

TIME-COURSE OF THE ENZYMATIC REDUCTION OF AZO-BODIPY 5i
The reduction process was carried out in the presence of the reducing cofactor, NADH, and monitored by the decrease in the absorbance at 615 nm for 5i. The immobilized enzyme and the azocompound 5i are incubated in 50% ethanol in 50 mM TRIS buffer at pH 7.0 in a spectrophotometric cell with stirring. Without the NADH, the absorbance at 615 nm (0.3) remains unaltered for 20 minutes, and after addition of reduced NADH the absorbance decreases down to 0.001 in 4 minutes ( Figure S16). At this moment, the reaction mixture exhibits a pink fluorescence under a deuterium lamp. In the absence of enzyme, the addition of NADH to the azocompound does not modify the absorbance at 615 nm and the azocompound remains unmodified.  ). DMEM supplemented with FCS and antibiotics will be referred to as complete medium. Cells were grown in a MIDI40 cell incubator (Thermo Scientific), with a 5% CO2 atmosphere, a 95% relative humidity and a constant temperature of 37 ºC. For the photocytotoxicity experiments, cells were plated on 24 wells plates and for fluorescence experiments, cells were plated onto round coverslips placed into wells.

Administration of compounds
Stock solutions of the corresponding compounds (5i or 6i) were prepared in DMSO (Panreac) at a concentration of 1 mg/mL (2 mM and 2.7 mM respectively). The work solutions were obtained by dissolving the compounds in complete medium. The final concentration of DMSO was always lower than 0.5% (v/v), and the lack of toxicity of this solvent for the cells was also tested and confirmed. All the treatments were performed when cultures reached around 60-70% of confluence.
For the time-course analyses of oxygen deprived conditions, HeLa cell grown on coverslips were treated with 5i (10 μM) for 2 h, place on a glass slide and visualized under fluorescent microscopy after 0, 10, 20 and 40 min.
To analyze the impact of azoreductase inhibitor, HeLa cells were incubated with DPI (10 μM in complete medium) and the azo 5i for a period of 2 h followed by 40 min of oxygen deprivation.
To carry out controls with HeLa cells incubated with 5i (10 μM) under normoxia conditions, they were cultured on 35mm petri dish with a 10mm coverslip inserted into the bottom (MatTek). The cells were analyzed under a confocal microscopy.

Intracellular localization.
HeLa cells were seeded on coverslips placed into 24-well plates at densities of 3 x 10 4 cells and allowed to grow for 48 h. After that, cells were incubated for 2 h in the presence of the corresponding azo-BODIPY 5i at 10 µM. The samples were washed twice with PBS, then mounted on slides with PBS and examined with a fluorescence microscope.

MTT viability assay
Cell viability were documented by the MTT assay. 26 Following appropriate treatments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well at a concentration of 0.5 ng/mL, and plates were incubated at 37 ºC for 2-3 h. The resulting formazan crystals were dissolved by the addition of DMSO and absorbance was measured at 540 nm. The results were expressed as cell survival percentage of control (cell survival (%) = (mean OD treated cells/mean OD value of control cells) x 100%).

Fluorescence Microscopy
Epifluorescence microscope Olympus BX61: Microscopic observations and photographs were performed in an Olympus photomicroscope IMT-2, equipped with a HBO 100 W mercury lamp and the corresponding filter sets for fluorescence microscopy: UV (365 nm), blue (450-490 nm, exciting filter BP 490) and green (545 nm, exciting filter BP 545) and a 100 x oil objective lens. The cell images were taken with a digital Camera: Olympus DP70; Fluorescence Filter Cube: U-MWIG; Exciter Filter: BP520-550 nm; Dichroic Beam Splitter: DM565 nm; Barrier Filter: BA580-IF.

Quantification of the fluorescence of the images
To carry out the quantification of the fluorescence of the images, the following steps have been carried out with FIJI (version: 2.3.0/1.53f): 1. Calibration of the images to obtain the area in SI units (microns). 2. Isolation of the channel of interest (in our case red) (the original image is in RGB). 3. Subtract a common background value (specifically 3.5) obtained from the average of several regions of each times. 4. Selection of a specific signal by applying a threshold following the "Moments" algorithm since it adjusts correctly to the desired signal. 5. Measure of the specific signal using this algorithm.

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The results represented in Figure S18 A corresponds to fluorescence images of HeLa cells (Figure S18 B) incubated with 5i (+ AZO) and subjected to different oxygen deprivation times (0, 10, 20, 30, 40, 50 and 60 min). Each bar in Figure S18 (A) corresponds to the mean fluorescence intensity of three different fields from a single experiment. As can be seen in Figure S18 (A) the change in fluorescence increased 8.3 times from 0 min to 60 min of anoxia.